Infection with the Hepatitis C virus (HCV) represents a serious world-wide health crisis. In more than 70% of infected individuals, the virus evades clearance by the immune system leading to a persistent HCV infection. The long term effects of persistent HCV infection range from an apparently healthy carrier state to chronic hepatitis, liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma. HCV is a leading cause of chronic liver disease. A leading therapy currently available for treatment of HCV infection uses a combination of pegylated α-interferon and ribavirin. However, many of the patients treated with this therapy fail to show a sufficient antiviral response. Additionally, interferon treatment also induces severe side-effects (i.e. retinopathy, thyroiditis, acute pancreatitis, depression) that diminish the quality of life of treated patients. Thus, it is important that more effective treatments be identified.
The identification of inhibitors of HCV replication and/or proliferation has been facilitated by the development of a cell-based system to study HCV replication. Inhibition of HCV replication may be performed using the HCV Replicon Assay developed in the laboratories of Bartenschlager (Lohman et al, Science 285, 110-113, 1999), Rice (Blight et al, Science 290, 1972-1974, 2000) and Lemon (Yi and Lemon, J. Virol. 78(15), 7904-7915, 2004). The assay is performed using the either the Huh-Luc-Neo cell line (HCV genotype 1b replicon; Lohman et al, Science 285, 110-113, 1999) or the En5-3/Htat2ANeo cell line (HCV genotype 1a replicon; Yi and Lemon, J. Virol. 78(15), 7904-7915, 2004) Huh-Luc-Neo cells are a human hepatoma cell line (Huh-7) stably expressing a bi-cistronic subgenomic HCV genotype 1b replicon (luc-neo/ET) containing the HCV IRES in which the structural protein sequences of HCV have been deleted and replaced by a construct containing sequences coding for the firefly luciferase reporter gene, the neomycin selectable marker and the EMCV IRES to direct expression of a truncated HCV genome expressing the genotype 1b non-structural proteins NS3, NS4A, NS4B, NS5A, and NS5B. The En5-3/Htat2ANeo cells are a Huh-7 cell line stably expressing the pLTR-SEAP (HIV LTR driven secreted alkaline phosphatase reporter) and a bi-cistronic subgenomic HCV genotype 1a replicon (Htat2ANeo) and is similar to the genotype 1b luc-neo/ET replicon except that the HCV structural proteins sequences have been replaced by the HIV tat gene, the FMDV 2A proteinase sequences, the neomycin selectable marker and the EMCV IRES which directs expression of the genotype 1a non-structural proteins (NS3-NS5B; Yi et al, Virology, 304(2), 97-210, 2002).
Strategies in new drug discovery often look to natural products for leads in finding new chemical compounds with therapeutic properties. One of the recurring problems in drug discovery is the availability of organic compounds derived from natural sources. Techniques employing combinatorial chemistry attempt to overcome this problem by allowing the high throughput synthesis and testing of hundreds or thousands of related synthetic compounds, called a chemical library. In designing the synthesis of a prospective therapeutic compound or a chemical library, one often looks to natural chemical motifs which are known to have broad biological activity.
Pyrazole has a long history of application in pharmaceutical and agrochemical industry, and posses a widespread occurrence as sub-structures in a large variety of compounds, which exhibit important biological activities and pharmacological properties. Elguero, J. In Comprehensive Heterocyclic Chemistry II; Katritzky, A. R.; Rees, C. W.; Scriven, E. F. V.; Pergamon-Elsevier Science: Oxiford, 1996; Vol. 6, pp. 1-75; (b) Sutharchanadevi, M.; Murugan, R. In Comprehensive Heterocyclic Chemistry II; Katritzky, A. R.; Rees, C. W.; Scriven, E. F. V.; Pergamon-Elsevier Science: Oxiford, 1996; Vol. 6, pp. 221-260.